(v?  /CO 


CHEMISTRY  OF  FLESH 

FURTHER  STUDIES  ON  THE  APPLICATION  OF  FOLIN’S 
CREATIN  AND  CREATININ  METHOD  TO  MEATS 
AND  MEAT  EXTRACTS. 


BY 

A.  D.  EMMETT  AND  H.  S.  GRINDLEY. 


(From  the  Laboratory  of  Physiological  Chemistry,  Department  of  Animal  Husbandry, 

University  of  Illinois.) 


From 


THE  JOURNAL  OF  BIOLOGICAL  CHEMISTRY. 
Vol.  EH,  No.  6,  November,  1907 


ttok-obYt 


E.  YO  (c  A c_^\ 

'rr\ 


CHEMISTRY  OF  FLESH. 

(sixth  paper)1 

FURTHER  STUDIES  ON  THE  APPLICATION  OF  FOLIN’S  CREATIN  AND 
CREATININ  METHOD  TO  MEATS  AND  MEAT  EXTRACTS. 

By  A.  D.  EMMETT  and  H.  S.  GRINDLEY. 

From  the  Laboratory  of  Physiological  Chemistry , Department  of  Animal 
Husbandry , University  of  Illinois. 

(Received  for  publication,  October  15,  1907.) 

Since  the  publication  of  the  first  paper2  from  this  laboratory 
upon  the  determination  of  creatin  and  creatinin  in  meats  and 
meat  extracts  further  studies  have  been  made  to  test  the  accuracy 
of  the  method  formerly  used.  This  additional  work  has  been  in 
part  prompted  by  the  paper  of  Otto  Hehner,3  who  takes  the  atti- 
tude that  Folin’s  creatin  method  if  directly  applied  to  commercial 
meat  extracts  does  not  give  accurate  results.  Hehner’s  chief 
criticism  is  that  15  cc.  of  the  1.2  per  cent  picric  acid  solution  used 
in  Folin’s  method  as  applied  to  the  determination  of  creatin  in 
urine  is  not  sufficient  for  the  determination  of  this  constituent 
in  commercial  meat  extracts.  In  his  article  Hehner  states  that 
in  such  cases  an  increased  quantity  of  the  picric  acid  solution 
should  be  used  and  he  recommends  the  use  of  a total  of  2 5 cc.  of  a 
1. 01  per  cent  solution  of  picric  acid  together  with  “a  quite  small 
amount  of  alkali,”  and  he  says  further  that  “ an  excessive  quantity 
of  the  alkali  diminishes  the  color.  ’ ’ This  difference  in  the  quantity 
and  strength  of  the  picric  acid  which  is  represented  by  6.1  cc.  of 
a 1.2  per  cent  solution,  gave  results  according  to  Hehner  which 
showed  commercial  meat  extracts  (Lemco,  Lazenty’s,  Armour’s 

1 H.  S.  Grindley:  Journ.  of  the  Amer.  Chem.  Soc.,  xxvi,  p.  1086,  1904; 
H.  S.  Grindley  and  A.  D.  Emmett:  Ibid.,  xxvii,  p.  658,  1905;  A.  D.  Em- 
mett and  H.  S.  Grindley:  Ibid.,  xxviii,  p.  25,  1905;  P.  F.  Towbridge  and 
H.  S.  Grindley \lbid.,  xxviii,  p.  469,  1906;  H.  S.  Grindley  and  H.  S.  Woods: 
This  Journal  ii,  p.  309,  1907. 

2 Grindley  and  Woods:  This  Journal,  ii,  p.  309,  1907. 

3 P harm.  Journ.,  lxxviii,  p.  683,  1907. 

491 


? 


492 


Chemistry  of  Flesh 


Baron  Liebig’s  and  Army  and  Navy)  to  contain  io  to  12  percent 
of  combined  creatin  and  creatinin,  whereas  Bauer  and  Barschall,1 
and  Grindley  and  Woods2  found  by  using  Folin’s  proportion  of  the 
reagents  4 per  cent  and  1 to  6.5  per  cents,  respectively,  of  these 
combined  extractives.  Such  differences  in  the  quantities  of 
creatin  and  creatinin  thus  obtained  in  meat  extracts  by  different 
analysts  certainly  demand  thorough  investigation,  if  this  con- 
venient method  of  determining  creatin  and  creatinin  in  such 
preparations  is  to  be  retained.  In  this  connection  it  should  be 
said,  that  Hehner  gives  none  of  his  detailed  analytical  data  and 
the  description  of  his  analytical  procedure  is  so  meager  that  it  is 
impossible  to  accurately  decide  the  details  of  his  method. 

Further,  as  a result  of  the  work  already  published  by  Folin3  and 
by  Benedict  and  Meyers4  in  testing  the  method  with  pure  creatin, 
Hehner’ s criticism  would  at  first  sight  seem  to  be  almost  without 
any  foundation.  On  the  other  hand,  the  extent  to  which  this 
method  of  determining  creatin  and  creatinin  is  already  being 
used  both  in  commercial  and  scientific  work  demands  that  the 
conditions  under  which  it  gives  accurate  results  should  be  fully 
determined  experimentally  beyond  any  reasonable  doubt.  With 
this  object  in  view,  the  work  here  reported  was  undertaken. 

Folin,5  in  first  presenting  this  method  for  the  quantitative 
determination  of  creatinin  and  creatin  in  urine,  recommended  the 
use  of  15  cc.  of  a 1.2  per  cent  solution  of  picric  acid  and  5 cc.  of  a 
10  per  cent  solution  of  sodium  hydroxide.  Later,6  in  modifying 
his  method  he  increased  the  amount  of  alkali  from  5 cc.  to  9 cc. 
on  account  of  the  increased  quantity  of  normal  hydrochloric  acid 
he  found  desirable  to  use  in  changing  the  creatin  to  creatinin. 
Bauer  and  Barschall,7  in  their  work  upon  beef  extracts,  used  15  cc. 
of  the  picric  acid  solution  and  5 cc.  of  the  alkali  solution.  Bene- 
dict and  Meyers,8  in  using  the  method  with  urine  and  with  beef 

1 Arbciten  aus  dem  kaiserlichen  Gesundheitsamte , xxiv,  p.  552. 

2 Loc.  cit. 

3 Zeitschr.  f.  physiol.  Chem.,  xli,  p.  223,  1904;  Festschrift  f.  Olaf  Ham- 
mar sten,  iii,  1906. 

* Amer.  Journ.  of  Physiol.,  xviii,  p.  4,  1907. 

5 Zeitschr.  f.  physiol.  Chem.,  xli,  p.  223,  1904. 

6 Festschrift  f.  Olaf  Hammarsten,  iii,  1906. 

7 Arbeiten  aus  dem  kaiserlichen  Gesundheitsamte , xxiv,  p.  552. 

8 Loc.  cit. 


A.  D.  Emmett  and  H.  S.  Grindley 


493 


extracts,  took  1 5 cc.  of  the  saturated  picric  acid  solution  and  10  cc. 
of  the  alkali  solution.  In  the  previous  publication1  upon  this 
subject  from  this  laboratory,  1 5 cc.  of  the  saturated  (1.2  per  cent) 
solution  of  picric  acid  and  5 cc.  of  the  10  per  cent  sodium  hydrox- 
ide solution  were  taken.  In  the  work  of  this  laboratory  the 
details  of  the  method  as  perfected  and  thoroughly  tested  by 
Folin  for  the  determination  of  creatinin  and  creatin  in  urine  were 
followed  as  closely  as  possible.  Further,  in  the  previous  com- 
munication, it  was  clearly  stated  that  the  color  of  the  unknown 
creatinin  picrate  solution  was  compared  with  8.0  mm.  of  the  f 
solution  of  potassium  bichromate  (24.54  grams  per  liter)  and 
not  with  the  color  obtained  by  allowing  the  picric  acid  and  alkali 
to  act  upon  a standard  creatinin  solution  as  Hehner  states  as 
the  method  employed  by  us.  In  all  our  work  we  have  accepted 
the  formulas  and  equivalents,  which  were  so  carefully  worked  out 
by  Folin. 

EXPERIMENTAL. 

In  testing  the  method,  the  influence  of  varying  the  quantity 
of  both  the  picric  acid  solution  and  the  alkali  solution  was  con- 
sidered. The  differences  in  the  intensity  of  the  color,  if  there  be 
any  within  narrow  limits,  due  to  the  length  of  time  the  solutions 
were  allowed  to  stand  were  not  taken  up  sufficiently  in  connection 
with  the  present  study  to  warrant  consideration  in  this  paper. 
In  all  the  determinations  herein  reported,  the  time  allowed  for 
the  development  of  the  color  was  as  nearly  as  possible  five  min- 
utes. The  purity  and  strength  of  the  picric  acid  solution  were 
thoroughly  tested.  The  picric  acid  was  found  to  be  quite  pure 
and  the  strength  of  the  picric  acid  solution  used  in  this  work  was 
1.2  per  cent.  A 10  per  cent  solution  of  sodium  hydroxide  was 
used  as  the  alkali.  The  volurrfes  of  the  1.2  per  cent  solution  of 
the  picric  acid  used  were  15,  30  and  45  cc.,  and  5,  10  and  15  cc. 
of  the  10  per  cent  solution  of  the  sodium  hydroxide  were  taken. 
All  the  readings  of  the  colorimeter  were  made  by  at  least  two 
persons  each  working  independently  of  the  other.  In  making 
the  observations  each  person  recorded  three  or  four  of  those 
agreeing  most  closely.  One  of  the  analysts  in  making  the  read- 


1 This  Journal,  ii,  p.  309,  1906. 


494 


Chemistry  of  Flesh 


ings  was  not  informed  as  to  the  nature  of  the  solutions  under 
examination.  The  average  of  the  readings  of  the  two  analysts 
was  taken  as  the  true  value  for  the  data  reported  in  the  tables 
here  given.  In  cases  where  the  colorimeter  was  used  continu- 
ously for  a considerable  length  of  time,  the  standard  bichromate 
solution  was  renewed  once  or  twice  to  avoid  any  possible  error  due 
to  evaporation. 

Detailed  description  of  the  method  used.  About  io  grams  of  the 
commercial  meat  extract  were  dissolved  in  water  and  the  result- 
ing solution  was  diluted  to  exactly  500  cc.  and  thoroughly 
mixed.  In  some  cases  the  solutions  thus  prepared  were  filtered 
through  dry  filters  while  in  other  cases  the  original  solutions 
were  not  filtered  before  being  used  for  the  determination  of 
the  creatinin  and  the  creatin.  For  the  determination  of  the 
creatinin,  aliquot  portions  of  the  sample  solution  which  would 
give  a reading  of  7 to  9 mm.  in  the  Duboscq  colorimeter  were 
transferred  to  500  cc.  measuring  flasks.  To  these  portions,  the 
measured  amounts  of  the  1.2  per  cent  picric  acid  solution  and 
the  10  per  cent  solution  of  sodium  hydroxide  were  each  added. 
The  mixture  was  then  shaken  and  allowed  to  stand  exactly  five 
minutes  when  it  was  immediately  and  quickly  diluted  to  the  500 
cc.  mark  with  distilled  water  and  thoroughly  mixed.  Readings 
upon  this  solution  were  made  at  once  in  the  colorimeter,  compar- 
ing the  depth  of  color  of  the  solution  to  be  tested  with  that  of 
a standard  half-normal  bichromate  (24.54  grams  per  liter)  solu- 
tion set  at  8 mm. 

For  the  determination  of  the  creatin  measured  portions  of  the 
sample  solution  were  transferred  to  small  Jena  beakers.  In  case 
the  volumes  of  the  solution  taken  amounted  to  more  than  10  cc., 
they  were  evaporated  upon  the  steam  bath  to  this  volume  and 
then  treated  with  10  cc.  of  normal  hydrochloric  acid.  The  crea- 
tin of  the  solutions  was  changed  to  creatinin  by  the  autoclave 
method  of  Benedict  and  Meyers1  which  has  proved  to  be  con- 
venient and  accurate.  To  do  this,  the  beakers  containing  the 
solutions  were  placed  in  an  autoclave  and  heated  at  a temperature 
of  1 1 70  C.-1200  C.  for  30  minutes.  After  these  solutions  were 
taken  from  the  autoclave  they  were  diluted  to  a definite  volume 

1 Amer.  Journ.  of  Physiol .,  xviii,  p.  398,  1907. 


A.  D.  Emmett  and  H.  S.  Grindley 


495 


and  measured  amounts  of  the  diluted  solutions  which  would 
give  a reading  of  7 to  9 mm.  in  the  colorimeter  were  transferred 
to  500  cc.  measuring  flasks.  The  details  of  procedure  were  then 
continued  exactly  as  directed  above  for  the  estimation  of  the 
creatinin.  The  data  of  the  experiments  are  given  below  in  full. 


Experiment  1.  For  this  experiment  9.2899  grams  of  a well-known 
brand  of  commercial  beef  extract  were  dissolved  in  water  and  the  result- 
ing solution  was  diluted  to  500  cc.  and  thoroughly  mixed.  The  solution 
was  then  filtered  through  dry  filters  and  portions  of  25  cc.  each  were  taken 
for  the  determination  of  creatinin  by  the  method  described  in  detail  above. 
The  results  of  the  determination,  in  which  varying  amounts  of  the  picric 
acid  and  the  sodium  hydroxide  solutions  were  used  for  the  same  volume, 
of  the  sample  solutions  are  given  in  the  following  table: 


TABLE  1.  CREATININ  RESULTS  OBTAINED  IN  EXPERIMENT  1. 


o 

METHOD. 

U 

O 

o 

"cd 

s 

o 

o 

l 

2 

o 

A 

c3 

rl 

SAMPLE. 

Picric  acid 
used. 

Alkali 

used. 

Reading  of 
imeter. 

o 

rj 

.a>-§ 

55  .S 
£ 

Total  weigl 
creatinin. 

O 

a . 

a 
u G 

Ph 

Beef  extract 

cc. 

cc. 

mm. 

mgr. 

mgr. 

p.  ct. 

2278a 

15 

5 

9.0 

9.000 

180.00 

1.94 

2278a 

“ “ 

15 

10 

8.9 

9.101 

182.02 

1.96 

2278a 

“ “ 

30 

5 

8.5 

9.529 

190.58 

2.05 

2278a 

U U 

30 

5 

8.9 

9.101 

182.02 

1.96 

2278a 

u u 

30 

10 

8.4 

9.643 

192.86 

2.08 

2278a 

u u 

30 

10 

8.2 

9.878 

197.56 

2.13 

2278a 

u a 

45 

5 

9.0 

9.000 

180.00 

1.94 

2278a 

u u 

Average  

45 

10 

8.9 

9.101 

182.02 

1.96 

2.00 

496 


Chemistry  of  Flesh 


Experiment  2.  For  this  experiment  the  same  filtered  solution  of  beef 
extract  was  taken  for  the  determination  of  creatin  as  was  used  above  in 
Experiment  1 for  the  determination  of  creatinin.  For  the  estimation  of 
the  creatin  in  the  solution,  five  portions  of  25  cc.  each  of  the  original 
filtered  solution  were  measured  out  and  the  creatin  which  they  contained 
was  converted  into  creatinin  in  the  autoclave  as  described  above.  The 
five  solutions  representing  125  cc.  of  the  original  solution  of  the  extract 
after  removal  from  the  autoclave  were  united  and  diluted  to  500  cc. 
Fifty  cubic  centimeter  portions  of  this  diluted  solution  were  taken  for 
the  determination  of  creatinin  by  the  method  described  in  full  above. 
The  analytical  results  obtained,  where  varying  amounts  of  the  picric 
acid  solution  and  the  sodium  hydroxide  solution  were  used  for  the  same 
volume  of  the  sample  solution,  are  given  in  the  following  table: 

TABLE  2.  CREATIN  RESULTS  OBTAINED  IN  EXPERIMENT  2. 


6 

METHOD. 

o 

© 

ORIGINAL  CREATININ 
PLUS  CREATININ  DUE 
TO  CREATIN. 

a 

;§> . 
O-S 

© 

a o^-n 
•rs  O 

ta  22th 

rQ 

iJ 

SAMPLE. 

Picric  acid 
used. 

Alkali 

1 used. 

Reading  of 
imeter. 

Original 

weight. 

Total 

weight. 

Per  cent. 

Z a 1 
o*-jg 

a 2 

g s 

p-i 

o § 

^ . 
g.a.s 

cm 

-.SX 

|.S.g 

i §1 

o,w© 

Ph 

2278a 

Beef  extract 

cc. 

15 

cc. 

5 

mm. 

16.1 

mgr. 

5.032 

mgr. 

201.28 

p.  ct. 

2.17 

p.  ct. 
2.00 

p.  ct. 

0.17 

p.  ct. 
0.20 

2278a 

“ 

u 

15 

10 

10.5 

7.714 

308.56 

3.32 

2.00 

1.32 

1.53 

2278a 

“ 

u 

15 

15 

9.6 

8.438 

337.52 

3.63 

2.00 

1.63 

1.89 

2278a 

“ 

u 

30 

10 

9.0 

9.000 

360.00 

3.88 

2.00 

1.88 

2.18 

2278a 

“ 

u 

30 

10 

9.1 

8.901 

356.04 

3.83 

2.00 

1.83 

2.12 

2278a 

a 

a 

30 

15 

9.0 

9.000 

360.00 

3.88 

2.00 

1.88 

2.18 

2278a 

“ 

“ 

30 

15 

9.2 

8.804 

352.16 

3.79 

2.00 

1.79 

2.08 

2278a 

u 

“ 

45 

10 

8.9 

9.101 

364.04 

3.92 

2.00 

1.92 

2.23 

2278a 

u 

11  

45 

10 

9.0 

9.000 

360.00 

3.88 

2.00 

1.88 

2.18 

A.  D.  Emmett  and  H.  S.  Grindley 


497 


Experiment  3.  For  this  experiment  two  solutions  were  prepared  from 
the  same  beef  extract  as  that  used  in  Experiments  Nos.  1 and  2.  In  the 
first,  namely,  No.  2278b,  11.3131  grams  of  the  extract  were  dissolved  in 
water  and  the  resulting  solution  was  diluted  to  500  cc.  and  thoroughly 
mixed.  In  the  second  solution,  No.  2278c,  10.3424  grams  of  the  extract 
were  dissolved  in  water  and  made  up  to  a definite  volume  of  500  cc. 
These  solutions  were  not  filtered  but  they  were  used  directly  for  the  work 
reported  in  this  and  the  following  experiment.  Portions  of  25  cc.  each 
of  these  unfiltered  solutions  were  taken  for  the  determination  of  creatinin. 

The  results  of  the  tests  are  given  in  the  following  table : 

TABLE  3.  CREATININ  RESULTS  OBTAINED  IN  EXPERIMENT  3. 


o 

METHOD. 

M 

o 

o 

c3 

o> 

Fh 

o 

o 

5 

"eg 

<o 

o 

£ 

A 

eg 

vA 

SAMPLE. 

Picric  acid 
used. 

Alkali 

used. 

Reading  of 
imeter. 

Weight  of 
inin. 

Total  weig' 
creatinin. 

o 

43 

Cl  . 

<u  c 

0.3 
u A 

O'- 

CM 

cc. 

cc. 

mm. 

mgr. 

mgr. 

p.  ct. 

2278b 

Beef  extract 

15 

5 

7.1 

11.408 

228.16 

2.02 

2278b 

a 

tt 

30 

5 

6.9 

11.739 

234.78 

2.08 

2278b 

u 

u 

30 

10 

6.9 

11.739 

234.78 

2.08 

2278b 

u 

u 

45 

5 

6.9 

11.739 

234.78 

2.08 

2278b 

u 

a 

Average 

45 

10 

6.9 

11.739 

234.78 

2.08 

2.07 

2278c 

Beef  extract 

15 

5 

7.8 

10.385 

207.70 

2.01 

2278c 

U 

U 

15 

5 

7.8 

10.385 

207.70 

2.01 

2278c 

“ 

u 

15 

10 

7.8 

10.385 

207.70 

2.01 

2278c 

“ 

“ 

15 

10 

7.9 

10.253 

205.06 

1.98 

2278c 

C( 

“ 

21 

10 

7.8 

10.385 

207.70 

2.01 

2278c 

C( 

u 

21 

10 

7.8 

10.385 

207.70 

2.01 

2278c 

(( 

a 

21 

15 

7.8 

10.385 

207.70 

2.01 

2278c 

“ 

a 

21 

15 

7.8 

10.385 

207.70 

2.01 

2278c 

a 

u 

30 

10 

7.8 

10.385 

207.70 

2.01 

2278c 

a 

u 

Average 

30 

10 

7.8 

10.385 

207.70 

2.01 

2.01 

498 


Chemistry  of  Flesh 


Experiment  4.  For  this  experiment  the  same  unfiltered  solution,  No. 
2278c,  was  taken  for  the  determination  of  creatin  as  was  used  above  for 
the  determination  of  creatinin  in  Experiment  3.  For  the  estimation  of 
the  creatin  four  portions  of  25  cc.  each  of  the  original  unfiltered  solution 
were  measured  out  and  the  creatin  which  they  contained  was  changed 
into  creatinin  as  usual.  The  four  solutions  representing  100  cc.  of  the 
original  solution  of  the  extract  after  removal  from  the  autoclave  were 
united  and  diluted  to  200  cc.  Twenty-five  cubic  centimeter  portions  of 
this  diluted  solution  were  taken  for  the  determination  of  creatinin  by  the 
usual  method.  The  results  of  the  tests  are  given  in  the  following  table: 


TABLE  4.  CREATIN  RESULTS  OBTAINED  IN  EXPERIMENT  4. 


Lab.  No. 

SAMPLE. 

METHOD. 

Reading  of  color- 
imeter. 

ORIGINAL  CREATININ 
PLUS  CREATININ 
DUE  TO  CREATIN. 

Per  cent  of  origin- 
al creatinin. 

Per  cent  of  creatin- 
in due  to  creatin. 

Per  cent  of  creatin 
(creatinin  due  to 
creatin  X 1.16). 

Picric  acid 
used. 

Alkali 

used. 

Original 

weight. 

Total 

weight. 

Per  cent. 

cc. 

cc. 

mm. 

mgr. 

mgr. 

p.  ct. 

p.  ct. 

p.  ct. 

p.  ct. 

2278b 

Beef  extract 

15 

10 

8.7 

9.310 

372.40 

3.60 

2.01 

1.59 

1.84 

2278b 

U U 

15 

10 

8.8 

9.205 

368.20 

3.56 

2.01 

1.55 

1.80 

2278b 

u u 

21 

10 

8.7 

9.310 

372.40 

3.60 

2.01 

1.59 

1.84 

2278b 

a « 

21 

10 

8.8 

9.205 

368.20 

3.56 

2.01 

1.55 

1.80 

2278b 

u u 

30 

10 

8.2 

9.878 

395.12 

3.82 

2.01 

1.81 

2.10 

2278b 

u u 

30 

10 

8.2 

9.878 

395.12 

3.82 

2.01 

1.81 

2.10 

A.  D.  Emmett  and  H.  S.  Grindley 


499 


Experiment  y.  For  this  experiment  a cold  water  extract  of  150.5287 
grams  of  lean  beef  round  was  prepared  by  the  methods  described  in  a 
former  paper.1  The  resulting  extract  was  made  up  to  a volume  of  7500 
cc.  Five  portions  of  1000  cc.  each  of  this  cold  water  extract  were  meas- 
ured out  and  evaporated  to  a volume  of  about  50  cc.  The  coagu- 
lated proteid  resulting  was  removed  by  filtration  and  thoroughly  washed 
with  hot  water.  The  filtrates  were  now  evaporated  to  a volume  of  1 o cc. 
and  the  creatin2  which  they  contained  was  converted  into  creatinin  by 
use  of  the  autoclave.  The  five  solutions  representing  five  liters  of  the 
original  cold  water  extract  after  removal  from  the  autoclave  were  united 
and  diluted  to  500  cc.  Fifteen  cubic  centimeter  portions  of  this  last  solu- 
tion were  taken  for  the  determination  of  creatinin  by  the  usual  method. 
The  results  of  the  tests  are  given  in  the  following  table: 


TABLE  5.  CREATIN  RESULTS  OBTAINED  IN  EXPERIMENT  5. 


6 

METHOD. 

JD 

O 

O 

A «i 

S g 

g 0 

0 0 

*3^ 

oX 

© 

O 

Lab.  N. 

SAMPLE. 

Picric  acid 
used. 

Alkali 

used. 

Reading  of 
imeter. 

0 

_ n 

.Mfl  fl* 

0)  fH.rt 

Total  weig 
creatinin 

Total  weig 
creatin.  1 
1.16.) 

1 Per  cent  0 

| atin. 

2279 

Lean  beef  round 

cc. 

15 

cc. 

5 

mm. 

7.3 

mgr. 

11.096 

mgr. 

554.80 

mgr. 

643.57 

p.  ct. 

0.428 

2279 

“ 

cc 

15 

5 

7.7 

10.519 

525.95 

610.10 

0.405 

2279 

11 

cc 

15 

5 

7.4 

10.950 

547.50 

635. 10  0.422 

2279 

u 

CC 

15 

5 

7.5 

10.800 

540.00 

626.40 

0.416 

2279 

“ 

cc 

15 

5t 

7.7 

10.520 

526.00 

610.16 

0.405 

2279 

u 

cc 

15 

10 

7.5 

10.800 

540.00 

626.40 

0.416 

2279 

“ 

cc 

15 

10* 

7.8 

10.380 

519.00 

602 . 04 

0.400 

2279 

“ 

cc 

30 

10 

7.0 

11.570 

578.50 

671.06 

0.446 

2279 

“ 

cc 

30 

10 

7.0 

11.570 

578.50 

671.06 

0.446 

2279 

“ 

cc 

30 

lot 

7.2 

11.250 

562 . 50 

652 . 50 

0.434 

2279 

cc 

30 

15 

7.0 

11.570 

578.50 

671.06 

0.446 

2279 

“ 

“ 

30 

10 

7.0 

11.570 

578.50 

671.06 

0.446 

2279 

11 

" 

45 

15 

7.0 

11.570 

578.50 

671.06 

0.446 

* Also  added  10  cc.  y HC1. 
t Also  added  10  cc.  NaCl,  6 per  cent. 


1 H.  S.  Grindley  and  A.  D.  Emmett:  Journ.  of  the  Amer.  Chem.  Soc., 
xxvii,  p.  658,  1905. 

2 Former  experiments  made  in  this  laboratory  demonstrated  the  fact 
that  fresh  meats  contain  only  the  slightest  trace  of  creatinin  if  any  at  all. 


500 


Chemistry  of  Flesh 


Experiment  6.  For  this  experiment  five  different  brands  of  beef  ex- 
tracts were  taken.  They  were  given  Laboratory  Nos.  2306,  2307,  2308, 
2309  and  2310.  The  following  weights  of  these  extracts  were  respectively 
taken  for  the  work,  11.7990,  10. 1120,  9.5615,  11.2308,  and  9.6105  grams. 
Each  of  these  weighed  portions  was  dissolved  in  water  and  the  solutions 
thus  obtained  were  diluted  to  500  cc.  and  thoroughly  mixed.  The  result- 
ing solutions  were  not  filtered  but  they  were  used  directly  for  the  work 
reported  in  this  and  the  following  experiment.  The  portions  of  these 
diluted  solutions  which  were  taken  for  the  several  determinations  are 
given  below  in  the  table  giving  the  results  of  the  tests  of  this  experiment. 

TABLE  6.  CREATININ  RESULTS  OBTAINED  IN  EXPERIMENT  6. 


0 

a 

METHOD. 

l 

o 

o 

o 

G3 

0 

U 

0 

o 

5 

o3 

0 

Fh 

0 

Lab.  Ni 

SAMPLE. 

Original  v< 
taken. 

Picric  acid 
used. 

Alkali 

used. 

Reading  of 
imeter. 

o 

0.2 

£ 

Total  weig 

creatinin. 

O 

a . 
8.5 

Jh.S 

0 

Oh 

cc. 

cc. 

cc. 

mm. 

mgr. 

mgr. 

p.  ct. 

2306 

Beef  extract 

8.5 

15 

5 

7.5 

10.80 

635.25 

5.38 

2306 

U U 

8.5 

15 

10 

7.4 

10.95 

644 . 08 

5.47 

2306 

u u 

8.5 

30 

10 

7.2 

11.25 

661.73 

5.61 

2306 

A verage  

8.5 

30 

10 

7.3 

11.09 

652.31 

5.53 

5.4.5 

2307 

Beef  extract 

23.0 

15 

5 

8.0 

10.13 

220.26 

2.18 

2307 

it  it 

23.0 

15 

10 

7.8, 

10.38 

225.66 

2.23 

2307 

u cc 

Average  

23.0 

30 

10 

7.6 

10.66 

231.75 

2.29 

2.28 

2308 

Beef  extract 

50.0 

15 

10 

7.8 

10.39 

103.85 

1.09 

2308 

CC  CC 

50.0 

21 

10 

7.8 

10.39 

103.85 

1.09 

2308 

cc  cc 

50.0 

21 

10 

7.8 

10.39 

103.85 

1.09 

2308 

“ “ 

50.0 

30 

10 

7.8 

10.39 

103.85 

1.09 

2308 

Average  

50.0 

30 

10 

7.8 

10.39 

103.85 

1.09 

1.09 

2309 

Beef  extract 

13.0 

15 

5 

7.3 

11.096 

426.75 

3.80 

2309 

CC  it 

13.0 

15 

5 

7.3 

11.096 

426.75 

3.80 

2309 

u cc 

13.0 

15 

10 

7.2 

11.225 

432.68 

3.85 

2309 

cc  cc 

13.0 

15 

10 

7.0 

11.571 

445.02 

3.96 

2309 

cc  cc 

13.0 

30 

10 

7.0 

11.571 

445 . 02 

3.96 

2309 

cc  u 

12.5 

30 

10 

7.3 

11.096 

443 . 84 

3.95 

2309 

CC  cc 

13.0 

30 

10 

7.1 

11.408 

438.75 

3.91 

2309 

cc  cc 

13.0 

30 

10 

7.1 

11.408 

438.75 

3.91 

2309 

it  cc 

Average  

13.0 

30 

15 

7.1 

11.408 

438.75 

3.91 

8.89 

2310 

Beef  extract 

17.0 

15 

5 

7.0 

11.57 

340.27 

3.54 

2310 

CC  CC 

17.0 

15 

10 

7.0 

11.57 

340.27 

3.54 

2310 

cc  cc 

Average  

17.0 

30 

10 

6.9 

11.74 

344.09 

3.58 

3.55 

A.  D.  Emmett  and  H.  S.  Grindley 


5oi 

Experiment  7.  For  this  experiment  the  same  unfiltered  solutions  of 
the  five  beef  extracts  used  above  in  Experiment  6 were  taken  for  the 
determination  of  creatin.  For  the  estimation  of  creatin  Nos.  2306,  2307, 
2309  and  2310,  five  portions  of  25  cc.  each  were  measured  from  each  of 
the  four  solutions  of  the  extracts  and  the  creatin  which  they  contained 
was  converted  into  creatinin  as  usual.  The  five  solutions  representing 
125  cc.  of  the  original  solutions  of  each  of  the  extracts  after  removal  from 
the  autoclave  were  united  and  diluted  to  500  cc.  For  the  estimation  of 
creatin  in  No.  2308  four  portions  of  25  cc.  each  were  measured  from  the 
original  solution  of  this  extract  and  the  creatin  contained  in  the  same 
was  converted  into  creatinin  as  usual.  The  four  solutions  representing 
100  cc.  of  the  original  solution  after  removal  from  the  autoclave  were 
united  and  diluted  to  200  cc.  The  portions  of  these  diluted  solutions 
which  were  taken  for  the  determination  of  the  creatinin  by  the  usual 
method  are  indicated  by  the  following  table  which  gives  the  detailed 
analytical  results  for  the  three  samples  of  beef  extract. 


TABLE  7.  CREATIN  RESULTS  OBTAINED  IN  EXPERIMENT  7. 


Lab.  No. 

SAMPLE. 

| Diluted  sol.  taken. 

METHOD. 

Reading  of  color- 
imeter. 

ORIGINAL  CREATININ 

PLUS  CREATININ  DUE 
TO  CREATIN. 

Per  cent  of  original 
creatinin. 

Per  cent  of  creat- 
inin due  to  crea- 
tin. 

Per  cent  of  creatin 

(creatinin  due  to 

creatin  X 1.16). 

Picric  acid 
used. 

Alkali 

used. 

Original 

weight. 

Total 

weight. 

Per  cent. 

cc. 

cc. 

cc. 

mm. 

mgr. 

mgr. 

p.  ct. 

p.  ct. 

p.  ct. 

p.  ct. 

2306 

Beef  extract.. 

50 

15 

5 

7.0 

11.571 

462 . 84 

3.92 

5.45 

2306 

“ 

u 

50 

15 

5 

7.1 

11.408 

456.32 

3.87 

5.45 

2306 

U 

u 

30 

30 

10 

7.0 

11.571 

771.79 

6.54 

5.451.09 

1.26 

2307 

« 

u 

50 

15 

5 

11.0 

7.364 

294.56 

2.91 

2.23 

0.68 

0.79 

2307 

U 

u 

70 

15 

10 

6.9 

11.594 

331.23 

3.28 

2.23 

1.05 

1.22 

2307 

U 

(( 

70 

30 

10 

6.4 

12.656 

361.58 

3.58 

2.23 

1.35 

1.57 

2308 

“ 

« 

60 

30 

10 

9.0 

9.000 

150.00 

1.57 

1.09 

0.48 

0.57 

2308 

C( 

u 

67 

30 

10 

8.3 

9.759 

145.70 

1.52 

1.09 

0.43 

0.50 

2309 

u 

u 

37 

15 

5 

9.1 

8.901 

481.10 

4.28 

3.89 

0.39 

0.45 

2309 

“ 

u 

37 

15 

5 

9.0 

9.000 

486.45 

4.33 

3.89 

0.44 

0.51 

2309 

u 

37 

15 

5 

8.9 

9.101 

491.91 

4.38 

3.89 

0.49 

0.57 

2309 

u 

u 

37 

15 

10 

8.0 

10.125 

547.26 

4.87 

3.89 

0.98 

1.14 

2309 

u 

u 

37 

15 

10 

7.9 

10.253 

554.18 

4.93 

3.89 

1.04 

1.21 

2309 

u 

“ 

37 

15 

10 

7.8 

10.385 

561.31 

5.00 

3.89 

1.11 

1.29 

2309 

u 

“ 

37 

30 

10 

7.7 

10.519 

568.55 

5.06 

3.89 

1.17 

1.36 

2309 

u 

a 

37 

30 

15 

7.9 

10.253 

554.18 

4.93 

3.89 

1.04 

1.21 

2310 

u 

u 

50 

15 

5 

8.1 

10.000 

400.00 

4.16 

3.54 

0.62 

0.72 

2310 

u 

“ 

50 

15 

10 

5.8 

13.966 

558.64 

5.81 

3.54 

2.27 

2.63 

2310 

u 

“ 

50 

30 

10 

5.6 

14.464 

578.56 

6.02 

3.54 

2.48 

2.88 

2310 

(( 

u 

35 

30 

10 

7.9 

10.253 

585.45 

6.09 

3.54 

2.55 

2.96 

5°2 


Chemistry  of  Flesh 


Experiment  8.  For  this  experiment  three  samples  of  urine  from  three 
different  persons  were  taken.  These  samples  were  given  Laboratory 
Nos.  2319,  2320  and  2321.  The  specific  gravities  of  the  urines  were 
respectively,  1.033,  1-029,  1.018.  In  each  sample  the  creatinin  was 
determined  by  the  usual  method,  using  however  different  quantities  of 
picric  acid  and  varying  quantities  of  alkali.  The  detailed  results  of  the 
experiment  are  given  in  the  table  that  follows: 


TABLE  8.  CREATININ  RESULTS  OBTAINED  IN  EXPERIMENT  8. 


© 

B 

B 

METHOD. 

color- 

"c8 

© 

o 

o 

A 

c3 

CD 

U 

o 

1 

Lab.  No. 

SAMPLE. 

Original  vc 
taken. 

Picric  acid 
used. 

1 Alkali 

used. 

Reading  of 
imeter. 

o 

CD. 5 
£ 

Total  weig 

creatinin 

o 

c 

g.g 

s-  .2 
© 

04 

cc. 

cc. 

cc. 

mm. 

mgr. 

mgr. 

p.  ct. 

2319 

Urine 

4.0 

15 

5 

6.9 

11.740 

205.45 

0.28 

2319 

a 

4.0 

15 

5 

7.0 

11.570 

202.48 

0.28 

2319 

(6 

4.0 

15 

10 

6.9 

11.740 

205 . 45 

0.28 

2319 

“ 

4.0 

30 

10 

6.4 

12.660 

221.55 

0.31 

2319 

Average 

4.0 

30 

10 

6.5 

12.460 

218.05 

0.30 

0.29 

2320 

Urine 

4.3 

15 

5 

7.8 

10.380 

142.41 

0.23 

2320 

Average 

4.3 

30 

10 

7.4 

10.950 

150.23 

0.25 

0.24 

2321 

Urine 

6.5 

15 

5 

7.4 

10.950 

176.78 

0 . ] 7 

2321 

U 

6.5 

15 

10 

7.3 

11.100 

179.20 

0.17 

2321 

U 

6.5 

15 

10 

7.5 

10.800 

174.42 

0.17 

2321 

u 

6.5 

30 

10 

7.3 

11.100 

179.20 

0.17 

2321 

u 

Average 

6.5 

30 

15 

7.3 

11.100 

179.20 

0.17 

0.17 

A.  D.  Emmett  and  H.  S.  Grindley 


503 


Experiment  g.  The  same  samples  of  urine  as  were  used  above  in 
Experiment  8 were  taken  for  the  determination  of  creatin.  For  the  esti- 
mation of  the  creatin  in  Laboratory  Nos.  2319  and  2321  four  portions  of 
10  cc.  each  were  measured  from  each  sample,  and  the  creatin  which  they 
contained  was  changed  into  creatinin  as  usual.  The  four  solutions  of 
each  sample  representing  40  cc.  of  the  original  urine  after  removal  from 
the  autoclave  were  united  and  diluted  to  250  cc.  The  portions  of  the 
diluted  solution  which  were  taken  for  the  determination  of  the  creatinin 
are  indicated  in  the  table.  For  the  estimation  of  creatin  in  Laboratory 
No.  2320  three  portions  of  10  cc.  each  were  taken  for  changing  the  creatin 
into  creatinin.  The  three  solutions  representing  30  cc.  of  the  original 
urine  after  removal  from  the  autoclave  were  united  and  diluted  to  250 
cc.  The  portions  taken  for  the  tests  are  indicated  in  the  table.  The 
complete  analytical  data  for  the  creatin  determination  in  the  three  sam- 
ples of  urine  are  given  in  detail  in  the  table  below: 


TABLE  9.  CREATIN  RESULTS  OBTAINED  IN  EXPERIMENT  9. 


o 

fc 

•s 

SAMPLE. 

Diluted  solution 
taken. 

METHOD. 

Reading  of  color- 
imeter. 

ORIGINAL  CREATININ 

PLUS  CREATININ  DUE 
TO  CREATIN. 

Per  cent  of  original 
creatinin. 

Picric  acid 
used. 

Alkali 

used. 

Original 

weight. 

Total 

weight. 

Per  cent. 

cc. 

cc. 

cc. 

mm. 

mgr. 

mgr. 

p.  ct. 

p.  ct. 

2319 

Urine 

20.0 

15 

5 

8.4 

9.643 

210.94 

0.29 

0.28 

2319 

20.0 

.15 

10 

8.2 

9.878 

216.08 

0.30 

0.28 

2319 

U 

20.0 

15 

10 

8.2 

9.878 

216.08 

0.30 

0.28 

2319 

“ 

20.0 

30 

30 

8.1 

10.000 

218.75 

0.30 

0.30 

2319 

“ 

25.0 

30 

10 

6.4 

12.656 

221.48 

0.31 

0.31 

2319 

u 

20.0 

30 

15 

8.3 

9.759 

213.48 

0.30 

2320 

u 

31.5 

15 

5 

8.8 

9.205 

143.69 

0.24 

0.24 

2320 

a 

31.5 

15 

10 

8.7 

9.310 

145.33 

0.24 

0.24 

2320 

u 

31.5 

15 

10 

8.8 

9.205 

143.69 

0.24 

0.24 

2320 

“ 

25.0 

30 

10 

10.0 

8.100 

159.33 

0.26 

0.24 

2320 

“ 

31.5 

30 

10 

8.5 

9.529 

148.75 

0.25 

0.24 

2320 

u 

31.5 

30 

10 

8.5 

9.529 

148.75 

0.25 

0.24 

2321 

u 

34.5 

15 

10 

8.8 

9.205 

175.08 

0 . 16 

0.17 

2321 

u 

34.5 

15 

10 

9.0 

9.000 

171.18 

0.16 

0.17 

2321 

u 

34.5 

30 

10 

8.8 

9.205 

175.08 

0.16 

0.17 

2321 

u 

34.5 

30 

10 

9.0 

9.000  171.18 

0.16 

0.17 

2321 

u 

25.0 

30 

1 

10 

10.9 

7.431  195.06 

0.18 

0.17 

504 


Chemistry  of  Flesh 


Experiment  io.  As  a further  check  on  this  method,  we  were  fortunate 
enough  to  obtain  through  the  kindness  of  Dr.  Folin  some  pure  creatin 
(Kahlbaum).  Crystallized  creatin  contains  one  molecule  of  water  of 
crystallization  and  in  our  sample  the  moisture  content  was  ascertained 
and  duly  allowed  for  in  the  subsequent  data  in  Table  io.  Several  por- 
tions of  the  sample,  given  Laboratory  No.  2289,  were  weighed  off  and 
each  dissolved  in  an  appropriate  quantity  of  distilled  water.  The  follow- 
ing weights  of  the  dry  substance  were  taken: 

(a)  . 1290  grams  (b)  .2217  grams  ( c ) .1165  grams 

(d)  .2705  “ ( e ) .2217  “ (/)  .4699  “ 

All  of  the  samples  were  dissolved  in  100  cc.  of  distilled  water,  except  in 
f,  which  was  dissolved  in  400  cc.  From  these  solutions,  five  10  cc.  portions 
of  samples,  b,  d,  e and  /,  and  nine  10  cc.  portions  of  samples  a and  c were 
transferred  to  small  beakers.  To  each  beaker  10  cc.  of  normal  hydro- 
chloric acid  was  added,  after  which  the  dehydration  was  carried  on  at  1 1 7- 
1 190  C.  in  the  autoclave  for  one-half  hour.  Each  of  the  resulting  creatinin 
solutions  of  the  respective  samples  was  transferred  to  a 500  cc.  measuring 
flask  and  diluted  to  the  mark.  After  thoroughly  mixing,  portions  of  50 
cc.  were  taken  for  the  usual  colorimetric  determinations.  Special  men- 
tion should  perhaps  be  made  at  this  point  for  subsequent  discussion  that 
solutions  a and  c contained  the  equivalent  of  90  cc.  of  normal  hydro- 
chloric acid  and  that  the  others,  b,  d,  e and  /,  contained  50  cc. 

The  following  table  gives  the  results  of  the  several  tests: 


TABLE  10.  SUMMARY  OF  RESULTS  OBTAINED  IN  EXPERIMENT  10. 


o 

>> 

0) 

B 

0 

s 

J3 

~o 

METHOD. 

o 

o 

creat- 
o ere- 

o-2 

0 

+=  3 

\ ix 

0 

hi  Pi 

g 

£ 

c3 

Weight  of  ( 
sample. 

"o 

_c 

[3) 

‘C 

O 

Diluted  v 
taken. 

Picric  acid. 

Alkali. 

[ Reading  of 
imeter. 

Weight  of 
inin  due  t 
atin. 

Total  weig 
creatinin 
creatin. 

Weight  of  c 
(Creatinii 
1.16) . 

O 

0) 

O 

0) 

Pk 

gms. 

cc. 

cc. 

cc. 

cc. 

mm. 

mgr. 

mgr. 

mgr. 

I p.  ct. 

2289A 

0.1290 

100 

50 

15 

\ 5 

22.6 

3.580 

39.82 

46.19 

35.81 

2289A 

0.1290 

100 

Average  ( 2 ) 

50 

15 

5 

22.6 

22.6 

3.580 

3.580 

39.82 

39.82 

46.1935.81 
4.6 . 19\35 .81 

2289A 

0.1290 

100 

50 

15- 

10 

9.1 

8.901 

98.89 

114.72 

88.93 

2289A 

0.1290 

100 

Average  (2) 

50 

1 

10 

9.1 

9.l\ 

8.901 

8.901 

98.89 

98.89 

114.72 

114.72 

88.93 

88.93 

2289A 

0.1290 

100 

50 

30 

10 

8.4 

9.643 

107.14 

124.29 

96.34 

2289A 

0.1290 

| 

100 

Average  ( 2 ) 

50 

30 

10 

8.5 

8.5 

9.529 

9.586 

105.88 

106.51 

122.82 

123.56 

95.21 

95.78 

2289A 

0.1290 

100 

50 

30 

15 

8.5i 

9.529 

105.88 

122.82 

95.21 

2289B 

0.2217 

100 

50 

15 

10 

9.5 

8.526 

170.52 

197.80 

89.21 

2289B 

0.2217 

100 

Average  ( 2 ) 

50 

15 

| 

10 

9.4 

9.5 

8.617 

8.572 

172.34 

171.43 

199.91 
198. 86\ 

90.17 

89.69 

A.  D.  Emmett  and  H.  S.  Grindley 


505 


Lab.  No. 

Weight  of  dry- 
sample. 

Original  volume. 

Diluted  volume 

taken. 

MET 

33 

g3 

.2 

*E 

0 

K 

'HOD. 

< 

Reading  of  color-  1 

imeter. 

Weight  of  ereat- 

inin  due  to  cre- 

atin. 

Total  weight  of 

creatinin  due  to 

creatin. 

Weight  of  creatin 

(.Creatinin  X 

1.16). 

Per  cent  of  creat  in. 

2289B 

0.2217 

100 

50 

30 

10 

9.0 

9.000 

180.00 

208.80 

94.18 

2289B 

0.2217 

100 

50 

30 

15 

9.0 

9.000 

180.00 

208.80 

94.18 

2289C 

0.1165 

100 

63 

15 

10 

9.0 

9.000 

89.29 

103.58 

88.91 

2289C 

0.1165 

100 

63 

15 

10 

8.8 

9.110 

90.37 

104.83 

89.99 

Average  (2) 

8.9 

9.055 

89.83 

104.21 

89.45 

2289C 

0.1165 

100 

63 

30 

10 

8.5 

9.529 

94.54 

109.66 

94.13 

2289C 

0.1165 

100 

63 

30 

10 

8.4 

9.643 

95.67 

110.97 

95.25 

Average  {2) 

8.5 

9.586 

95.11 

110.32 

94.69 

2289C 

0.1165 

100 

63 

30 

15 

8.5 

9.529 

94.54 

109.66 

94.13 

2289D 

0.2705 

100 

50 

15 

5 

9.0 

9.000 

180.00 

208.80 

76.89 

2289D 

0.2705 

100 

50 

15 

10 

7.7 

10.519 

210.38 

244.04 

90.21 

2289D 

0.2705 

100 

50 

15 

10 

7.6 

10.658 

213.16 

247.27 

91.04 

Average  ( 2 ) 

7.7 

10.589 

211.77 

245.65 

90.63 

2289D 

0.2705 

100 

50 

30 

10 

7.2 

11.250 

225. 10|261. 11 

96.49 

2289D 

0.2705 

100 

50 

30 

10 

7.3 

11.096 

221.92 

257.43 

95.17 

2289D 

0 . 2705 

100 

50 

30 

10 

7.3 

11.096 

221.92 

257.43 

95.17 

2289D 

0.2705 

100 

50 

30 

10 

7.4 

10.946 

218.92 

253.95 

93.88 

Average  (4) 

7.3 

11.097 

221 . 97 

257.48 

95.18 

2289D 

0.2705 

100 

50 

30 

15 

7.4 

10.946 

218.92 

253.95 

93.88 

2289E 

0.2217 

100 

50 

15 

5 

11.1 

7.300 

145.94 

169.29 

76.36 

2289E 

0.2217 

100 

50 

15 

10 

9.0 

9.000 

180.00 

208.80 

94.18 

2289E 

0.2217 

100 

50 

15 

10 

8.9 

9.101 

182.02 

211.14 

95.28 

Average  (2s) 

9.0 

9.051 

181.01 

209.97 

94.71 

2289E 

0.2217 

100 

50 

30 

10 

8.9 

9.101 

182.02 

211.14 

95.24 

2289E 

0.2217 

100 

50 

30 

10 

8.9 

9.101 

182.02 

211.14 

95.24 

2289E 

0.2217 

100 

50 

30 

10 

8.9 

9.101 

182.02 

211.14 

95.24 

Average  (3) 

8.9 

9.101 

182.02 

211.14 

95.24 

2289F 

0.4699 

200 

50 

15 

5 

10.2 

7.940 

317.64 

368.46 

78.47 

2289F 

0.4699 

200 

50 

15 

5 

10.4 

7.788 

311.52 

361.36 

76.88 

Average  (2s) 

10.3 

7.864 

314.58 

364.91 

77.68 

2289F 

0.4699 

200 

50 

15 

10 

8.4 

9.643 

385.72 

447.44 

95.22 

2289F 

0.4699 

200 

50 

15 

10 

8.3 

9.759 

390.36 

452 . 82 

96.38 

Average  (2) 

8.4 

9.701 

388.04 

450.13 

95.80 

2289F 

0.4699 

200 

50 

30 

10 

8.0 

10.125 

405.00l469.80 

99.98 

2289F 

0.4699 

200 

50 

30 

10 

8.0 

10.125 

405.00 

469.80 

99.98 

Average  (2) 

8.0 

10.125 

405.00 

469.80 

99.98 

2289F 

0.4699 

200 

50 

30 

15 

8.0 

10.125 

405.00 

469.80 

99.98 

5°6 


Chemistry  of  Flesh 


Experiment  n.  For  this  experiment  several  tests  were  made  upon 
solutions  of  creatinin  (Merck’s).  This  product  was  found  to  be  only  about 
85  per  cent  pure,  there  being  also  present  approximately  10  per  cent  of 
creatin  and  2.07  percent  of  moisture.  As  a result,  the  material  in  question 
could  not  be  used  to  serve  the  double  purpose  intended,  namely,  the  check- 
ing of  the  method  and  the  standardizing  of  the  bichromate  solution. 

However,  to  ascertain  the  variations,  if  any,  in  the  percentage  of 
creatinin  in  the  sample  when  different  proportions  of  the  picric  acid  and 
alkali  were  used  three  portions  were  weighed  out.  Sample  a weighed 
0.1286  grams  (dry);  b,  0.1127  grams  (dry),  and  c,  0.2055  grams  (dry). 
The  first  two  samples  were  dissolved  in  500  cc.  of  distilled  water  and  after 
thoroughly  mixing,  50  cc.  were  taken  for  the  usual  procedure.  Sample 
c was  dissolved  in  250  cc.  of  water  and  100  cc.  of  this  solution  were  taken 
and  diluted  to  500  cc.  after  which  75  cc.  were  used  for  the  regular  deter- 
mination. 

The  detailed  data  resulting  are  given  in  the  following  table : 


TABLE  11.  SUMMARY  OF  RESULTS  OBTAINED  IN  EXPERIMENT  11. 


Lab.  No. 

1 

Weight  of  sample. 

Original  volume. 

Original  or  diluted 
volume  taken. 

Picric  acid  g 

THOD. 

M 

< 

Reading  of  color- 
imeter. 

Weight  of  Creat- 
inin. 

Total  weight  of 
creatinin. 

firms. 

cc. 

cc. 

CC. 

CC. 

mm. 

mgr. 

mgr. 

2353a 

.1286 

500 

50 

15 

5 

8.7 

9.310 

93.10 

2353a 

.1286 

500 

50 

15 

5 

8.8 

9.205 

92.05 

2353a 

.1286 

500 

50 

15 

10 

7.6 

10.658 

106.58 

2353a 

.1286 

500 

50 

15 

10 

7.6 

10.658 

106.58 

2353a 

.1286 

500 

50 

30 

10 

7.4 

10.946 

109.46 

2353a 

. 1286 

500 

50 

30 

10 

7.5 

10.800 

108.00 

2353a 

.1286 

500 

50 

30 

15 

7.4 

10.946 

109.46 

2353a 

.1286 

500 

50 

30 

15 

7.5 

10.800 

108.00 

2353b 

.1127 

500 

50 

15 

5 

10.2 

7.941 

79.41 

2353b 

.1127 

500 

50 

15 

10 

8.8 

9.205 

92.05 

2353b 

.1127 

500 

50 

15 

10 

8.8 

9.205 

92.05 

2353b 

. 1127 

500 

50 

30 

10 

8.6 

9.415 

94.15 

2353b 

.1127 

500 

50 

30 

10 

8.7 

9.310 

93.10 

2353b 

.1127 

500 

50 

30 

15 

8.8 

9.205 

92.05 

2353c 

.2055 

250 

75 

15 

10 

9.0 

9.000 

150.03 

2353c 

.2055 

250 

75 

15 

10 

9.0 

9.000 

150.03 

2353c 

.2055 

250 

75 

30 

10 

8.0 

10.125 

168.78 

2353c 

.2055 

250 

75 

30 

10 

8.0 

10.125 

168.78 

2353c 

.2055 

250 

75 

30 

15 

8.0 

10.125 

168.78 

A.  D.  Emmett  and  H.  S.  Grindley 


507 


DISCUSSION  OF  RESULTS. 

It  is  quite  apparent  from  the  data  reported  in  the  preceding 
pages  that  the  Folin  method  of  estimating  creatinin  and  creatin, 
when  properly  modified,  is  as  applicable  to  meat  and  meat  extract 
as  it  is  to  urine.  Further,  it  is  evident  that  if  the  details  of  the 
method  are  properly  followed,  reliable  and  concordant  results 
can  be  obtained. 

Original  Creatinin.  (a)  Influence  of  alkali.  By  compar- 
ing the  data  in  Tables  1,  3,  6 and  8 which  relate  to  the  per- 
centage of  creatinin  in  meat  extracts  and  urine,  it  is  seen  that  in 
so  far  as  the  initial  quantity  of  creatinin  is  concerned,  the  influence 
of  increasing  the  amount  of  alkali  is  practically  nil.  If  15,  21  or 
30  cc.  of  picric  acid  of  1.2  per  cent  are  used  with  either  5,  10  or 
15  cc.  of  sodium  hydroxide  of  10  per  cent  strength  the  readings 
of  the  colorimeter  for  the  same  volume  of  the  solutions  under 
examination,  are  in  general  slightly  higher  for  the  5 cc.  quantities 
than  for  the  10  cc.  portions.  Stating  these  facts  in  terms  of 
percentages  of  creatinin,  the  10  and  15  cc.  quantities  of  sodium 
hydroxide,  which  are  approximately  8 and  13  cc.,  respectively, 
in  excess  of  the  necessary  amount  to  cause  neutralization  and 
solution  of  the  precipitate,  gave  but  very  slightly  higher  percent- 
ages than  did  the  5 cc.  quantities.  These  actual  differences  in 
the  case  of  meat  extracts  were  0.02  to  0.17  per  cent,  being  on  the 
average  0.05  per  cent.  In  the  case  of  the  creatinin  (Merck’s), 
which  as  previously  stated  contained  10  per  cent  of  creatin,  the 
data  in  Table  11,  show  the  variations  in  amount  of  alkali  to 
produce  an  appreciable  difference.  The  weights  of  original 
creatinin  resulting  from  the  use  of  10  or  15  cc.  of  alkali  are  the 
same,  but  for  those  resulting  from  the  use  of  5 cc.,  they  are  dis- 
tinctly lower,  being  in  the  former  cases  on  an  average  of  106.58 
mgms.,  and  in  the  latter  92.58  mgms.,  a difference  of  14.0 
mgms.  No  positive  explanation  can  be  given  for  this  large  varia- 
tion. It  is  easily  seen  that  the  differences  in  some  cases  in  the 
readings  due  to  the  amounts  of  alkali  might  be  from  2 to  4 mm. 
as  was  the  case  with  the  beef  extracts  and  urine,  and  this  would 
mean  with  such  a strong  solution,  a difference  of  2 to  4 per  cent. 

From  the  above  discussion,  the  data  show  that,  contrary  to 
Hehner’s  statement,  an  excess  of  alkali,  using  5,  10  and  15  cc.  of 


5°8 


Chemistry  of  Flesh 


a io  per  cent  solution,  does  not  diminish  the  depth  of  the  color 
produced  with  creatinin  picrate  but  rather  increases  it.  It  would 
seem,  therefore,  that  it  would  be  best  to  use  io  cc.  of  a io  per 
cent  solution  of  alkali  in  all  instances,  it  having  been  shown  that 
this  amount  of  alkali  has  no  detrimental  effect  whatever. 

(b)  Influence  of  picric  acid.  In  studying  the  data  in  Tables 
i,  3 and  6 on  beef  extract,  8 on  urine,  and  n on  pure  creatinin, 
it  will  be  seen  that  the  variations  in  the  percentages  of 
original  creatinin  are  extremely  slight  when  the  quantity  of 
picric  acid  is  increased  from  15  to  21,  30  or  45  cc.  in  the  cases 
where  10  cc.  of  the  alkali  are  used.  In  fact,  it  can  be  stated,  as 
far  as  the  creatinin  sample  and  also  the  meat  extract  samples 
are  concerned,  that  the  15  cc.  test  of  picric  acid  (1.2  per  cent) 
gave  as  high  a percentage  of  creatinin  as  did  the  21  cc.  test 
(which  is  the  equivalent  of  Hehner’s  25  cc.  portion,  1.01  per 
cent),  or  even  the  30  cc.  test.  This  fact  is  most  clearly  brought 
out  in  Tables  3 and  6 where  the  analysis  of  six  different 
samples  of  meat  extract  are  reported.  Sample  2306  has  a varia- 
tion, taking  the  average  of  the  readings  of  the  30  cc.  tests,  of 
0.10  per  cent  on  a total  of  5.47  per  cent  of  original  creatinin. 
Sample  2307  shows  an  increase  of  0.06  per  cent  for  the  30  cc.  of 
the  picric  acid  against  that  of  15  cc.  on  a total  of  2.23  per  cent. 
Similarly,  the  data  for  sample  2310  gave  a gain  of  0.04  per 
cent  for  the  30  cc.  of  picric  acid  when  compared  with  the  15  cc. 
on  a percentage  of  creatinin  of  3.55.  These  slight  differences  are 
no  greater  than  the  errors  which  might  be  due  to  the  matching 
of  the  color,  to  the  carrying  out  of  the  technique  of  the  method 
or  to  the  sampling  of  the  products. 

In  the  case  of  the  urine,  Table  8,  the  variations  in  the  readings 
for  sample  2319  are  greater  than  in  the  cases  just  considered 
but  those  for  sample  2321  show  the  same  variations  as  the  meat 
extracts.  When  calculated  to  the  percentage  of  creatinin  these 
differences  in  the  urine  are  insignificant.  Further,  when  the 
analysis  of  the  three  samples  of  the  pure  creatinin  are  con- 
sidered, it  is  seen  that  the  readings  of  the  two  tests,  15  and  30  cc. 
of  picric  acid  are  almost  identical.  These  small  differences  are 
no  greater  than  would  be  expected.  However,  if  these  values 
are  calculated  to  percentages,  the  variations  are  about  1.0  per 
cent.  The  cause  of  this  difference  is  not  to  be  found  as  Hehner 


A.  D.  Emmett  and  H.  S.  Grindley 


509 


states  in  the  greater  amount  of  creatinin  in  the  solution  which  is 
being  treated  with  picric  acid,  because  in  this,  and  the  previous 
work  of  this  laboratory,  whether  the  sample  under  examination 
was  meat  extract,  urine,  or  creatinin  itself,  such  an  aliquot  por- 
tion of  the  original  solution  was  taken  that  when  it  was  treated 
in  the  usual  manner  with  picric  acid  and  alkali,  and  diluted, 
the  resulting  color  gave  a reading  of  7 to  9 mm.  on  the  scale  of 
the  colorimeter  when  compared  with  the  standard  bichromate 
set  at  8 mm.  In  other  words,  all  the  solutions  tested  at  the  time 
of  the  reading  contained  approximately  the  same  quantity  of 
original  or  converted  creatinin.  The  real  cause  of  the  influence 
of  the  slight  differences  in  the  readings  upon  the  variations  cal- 
culated in  percentage,  naturally  lies  in  the  differences  in  the 
weights  of  the  substances  taken  for  the  samples,  since  the  pro- 
portion of  the  dilutions  were  approximately  the  same.  The 
weights  of  the  samples  were  always  about  150  grams  for  meats, 
10  grams  for  meat  extracts  and  0.12  grams  for  the  creatinin,  and, 
consequently,  it  is  plainly  seen  that  any  variation  in  the  readings 
would  be  very  much  less  apparent  in  the  percentage  composition 
in  the  case  of  the  meats  than  in  the  meat  extract  or  the  impure 
creatinin. 

From  this  discussion  upon  the  influence  of  varying  the  quantity 
of  picric  acid  in  cases  where  10  cc.  of  a 10  per  cent  solution  of 
alkali  were  used,  it  seems  safe  to  state  that  it  makes  no  difference, 
when  determining  the  original  creatinin,  whether  15,  21  or  30  cc. 
of  the  1.2  per  cent  solution  of  picric  acid  are  used.  The  final 
percentages  are  practically  identical. 

Creatinin  due  to  Creatin.  (a)  Influence  of  alkali.  In  the 
former  paper,  it  was  stated  that  the  creatin  in  the  samples  was 
changed  to  creatinin  by  using  25  cc.  of  Ty  hydrochloric  acid 
and  that  the  resulting  solutions  after  proper  dilution  were  treated 
with  15  cc.  of  picric  acid  (1.2  per  cent)  and  5 cc.  of  alkali  (10  per 
cent) . The  data  herein  reported  were  ascertained  by  using  10  cc. 
of  normal  hydrochloric  acid  for  the  dehydration  or  four  times  the 
amount  previously  taken.  Following  this  change  in  the  amount 
of  acid,  the  quantity  of  alkali  was  also  increased.  These  modifi- 
cations were  adopted  on  account  of  the  recommendations  of 
Folin  in  his  second  paper,1  that  in  the  determining  of  creatin  in 

1 Festschrift  f.  Olaf  Hammarsten , iii,  1906. 


Chemistry  of  Flesh 


5io 

abnormal  urine  10  cc.  of  normal  hydrochloric  acid,  15  cc.  of 
picric  acid  (1.2  per  cent)  and  9 cc.  of  sodium  hydroxide  (10  per 
cent)  should  be  taken. 

In  studying  the  influence  of  different  proportions  of  alkali 
when  varying  amounts  of  dehydrated  creatin  and  original  cre- 
atinin  were  present,  two  series  of  tests  were  carried  out.  In  the 
first,  5 and  10  cc.  of  the  alkali  were  used  against  15  cc.  of  the  picric 
acid,  and  in  the  second,  10  and  15  cc.  of  the  alkali  were  employed 
with  30  and  sometimes  45  cc.  of  the  picric  acid.  It  was  ascer- 
tained in  the  case  of  meat  extracts  that  approximately  1 to  2 cc. 
of  the  10  per  cent  alkali  were  sufficient  to  dissolve  the  precipitate 
and  to  produce  a red  coloration. 

The  data  in  Tables  2,  4 and  7,  relating  to  meat  extracts, 
show  the  effect  of  using  5 and  10  cc.  of  the  alkali  with  15  cc.  of 
the  picric  acid.  It  is  very  apparent  that  the  5 cc.  portion  is 
entirely  too  small.  This  fact  is  perhaps  shown  most  markedly 
in  sample  2306,  where  the  total  creatinin  is  3.90  per  cent  while 
that  for  the  original  creatinin  is  5.45  per  cent.  In  other  words, 
the  5 cc.  of  alkali  produced  a color  which,  when  compared  with 
the  standard,  represented  a value  of  29.4  per  cent  less  than  the 
amount  obtained  before  dehydration.  A comparison  of  the  5 
and  10  cc.  tests  is  best  brought  out  in  the  case  of  samples  2278a, 
Table  2;  and  2309  and  2310,  Table  7.  The  differences  in  the 
readings  with  the  two  quantities  of  alkali  vary  from  1.1  mm.  in 
2309  to  5.6  mm.  in  2278a.  These  data  when  calculated  to  the 
percentage  of  creatinin  as  creatin,  show  a range  of  0.7  to  1.33, 
respectively,  in  favor  of  the  10  cc.  portion  of  alkali. 

Very  little  can  be  said  regarding  the  influence  of  the  quantity 
of  alkali  in  the  case  of  the  meats  and  urines  since  the  data  are  too 
few  for  consideration.  The  slight  differences  in  those  instances 
that  are  reported  are  inappreciable  when  calculated  to  their 
final  percentages.  However,  in  connection  with  the  meat  extract 
the  facts  show  very  plainly  that  the  10  cc.  of  alkali  when  used 
with  15  cc.  of  picric  acid  gives  higher  results  than  the  5 cc.  portion. 

In  the  second  trial  where  10  and  15  cc.  of  alkali  were  used  with 
30  or  45  cc.  of  picric  acid,  the  resulting  data  for  the  beef  extracts 
and  meat  are  fairly  constant.  The  few  variations  amount  in 
the  maximum  to  only  0.2  mm.  This  would  indicate  that  the 
slight  differences  were  not  due  to  the  excess  of  alkali,  but  rather 


A.  D.  Emmett  and  H.  S.  Grindley  51 1 

to  errors  in  the  technique.  However,  it  should  perhaps  be 
stated  that  these  differences  are  in  the  main  in  favor  of  the  lower 
readings  for  the  10  cc.  test,  but  when  the  data  in  Table  10,  relat- 
ing to  pure  creatin,  are  also  taken  into  account  the  evidence  seems 
to  be  such  that  it  can  be  stated  that  there  are  no  differences 
resulting  in  using  either  10  or  15  cc.,  of  alkali  with  30  cc.  of  the 
picric  acid,  beyond  the  experimental  errors. 

From  the  above  consideration,  the  data  show  that  the  quantity 
of  alkali  does  influence  the  depth  of  color,  that  a small  quantity 
does  not  yield  as  high  a percentage  as  a large  one;  that  a large 
excess  does  not  give  low  results,  and  that  the  accepted  10  cc. 
portion,  yields  better  results  than  the  5 cc.  and  similar  results  to 
the  15  cc.  portion. 

(b)  Influence  of  picric  acid.  In  the  preceding  pages,  the  effect 
of  using  varying  amounts  of  alkali  and  picric  acid  has  been 
considered  where  preformed  creatinin  was  to  be  determined,  and 
in  the  above  paragraphs,  the  influence  of  different  quantities  of 
alkali  has  been  discussed  in  the  case  where  both  the  preformed 
and  dehydrated  creatinin  were  present.  It  is  the  purpose  of  this 
section  to  digest  the  data  herein  reported  which  refer  directly  to 
the  effect  of  using  larger  quantities  of  picric  acid  than  has  been 
customary  in  determining  creatin  as  creatinin. 

Hehner  noticed  in  his  work  that  an  increase  of  the  picric  acid 
from  15  cc.  to  25  cc.  (1.01  per  cent)  caused  a marked  difference  in 
the  amount  of  creatinin  found  to  be  present  in  meat  extracts. 
Instead  of  obtaining  with  25  cc.  of  picric  acid  (1.01  per  cent)  6 to  7 
per  cent  of  creatinin,  which  he  got  with  the  equivalent  of  15  cc. 
of  1.2  per  cent  acid,  he  reported  10  to  12  per  cent.  He  found, 
further,  that  a larger  amount  of  picric  acid  had  no  increased 
effect. 

In  our  work,  the  amounts  of  picric  acid  used  were  15,  21,  30 
and  45  cc.,  and  since  it  was  found  that  10  cc.  of  the  sodium 
hydroxide  worked  satisfactorily,  this  quantity  of  alkali  was 
taken  throughout  for  the  comparison.  The  data  in  Tables  2,4, 
and  7 on  meat  extracts,  Table  5 on  meat,  and  Table  10  on  pure 
creatin,  show  that  in  the  case  of  30  cc.  of  the  picric  acid  the  gen- 
eral tendency  is  to  produce  a lower  reading  and  hence  a higher 
percentage  of  creatinin  as  creatin.  These  differences  in  the  read- 
ings for  the  meat  extracts  are  practically  nothing  in  sample  2309 


512 


Chemistry  of  Flesh 


and  1.4,  0.6,  0.5  and  0.2  mm.  in  samples  2278a,  2278b,  2307  and 
2310,  respectively;  for  the  meat  they  are  0.5  mm.,  and  for  the 
pure  creatin  0.1  to  0.6  mm.  Concerning  the  data  for  the  urine, 
Table  9,  the  differences  due  to  the  increased  amount  of  picric 
acid  are  very  slight,  being  in  samples  2319  and  2321  almost 
nothing  and  in  sample  2320,  0.3  mm. 

In  general  then,  it  may  be  stated  for  the  meat  extracts,  meat 
and  pure  creatin  that  the  additional  quantity  of  picric  acid  may 
have  no  effect  in  some  cases  and  in  others  it  may  cause  a decrease 
in  the  readings  of  0.2  to  1.4  mm.  From  the  data  in  the  following 
Table  12,  which  gives  a summary  of  these  facts,  the  differences 


TABLE  12.  SUMMARY  OF  RESULTS  ON  INFLUENCE  OF  PICRIC  ACID. 

{Creatin.) 


n 

READINGS  OF  COLOR- 
IMETER. 

PERCENTAGE  OF  CREATIN. 

Lab.  N< 

SAMPLE. 

Picric  acid 
15  cc. 

Picric  acid 
30  cc. 

! 

Difference. 

Picric  acid 
15  cc. 

Picric  acid 

30  cc. 

Difference. 

2278a 

Beef  extract 

mm. 

10.5 

mm. 

9.1 

mm. 

1.4 

p.  ct. 

1.53 

p.  ct. 

2.15 

p.  ct. 
0.62 

2278c 

U (l 

8.8 

8.2 

0.6 

1.82 

2.10 

0.28 

2307 

u a 

6.9 

6.4 

0.5 

1.22 

1.57 

0.35 

2309 

u cc 

7.9 

7.8 

0.1 

1.29 

1.36 

0.07 

2310 

cc  cc 

5.8 

5.6 

0.2 

2.88 

2.96 

0.08 

2279 

Meat 

7.5 

7.0 

0.5 

0.42 

0.45 

0.03 

2319 

Urine 

8.2 

8.1 

0.1 

0.30 

0.30 

0.00 

2320 

cc 

8.8 

8.5 

0.3 

0.24 

0.25 

0.01 

2321 

cc 

8.9 

8.9 

0.0 

0.16 

0.16 

0.00 

2289a 

Pure  creatin 

9.1 

8.5 

0.6 

88.93 

95.78 

6.85 

2289b 

CC  CC 

9.5 

9.0 

0.5 

89.69 

94.18 

4.49 

2289c 

u cc 

8.9 

8.5 

0.4 

89.45 

94.69 

5.24 

2289d 

cc  cc 

7.7 

7.3 

0.4 

90.63 

95.18 

4.55 

2289e 

cc  cc 

9.0 

8.9 

0.1 

94.71 

95.24 

0.53 

2289f 

u cc 

8.4 

8.0 

0.4 

95.80 

99.98 

4.18 

in  the  percentage  of  creatin  in  the  meat  extracts  are  seen  to 
vary  from  0.07  to  0.62,  the  total  percentage  of  creatin  being  1.4 
to  3.0.  The  meat  shows  a corresponding  difference,  a gain  of 
0.03  per  cent  on  a total  of  0.446  per  cent.  In  the  case  of  the  urine 
the  variations  are  practically  nothing,  and  in  that  of  the  pure 


A.  D.  Emmett  and  H.  S.  Grindley  513 

creatin,  they  are  on  the  average  4.4  per  cent  greater  for  the  30 
cc.  portion  of  acid. 

A second  point  should  be  considered,  as  to  the  influence  of 
using  still  more  of  the  picric  acid.  The  data  for  the  sample  of 
beef  extract  2278a,  Table  2,  and  that  for  the  meat  2279,  Table  5, 
show  that  an  additional  15  cc.  of  acid,  or  45  cc.  in  all,  does  not 
cause  any  further  change  than  that  brought  about  by  the  30  cc. 
test. 

The  above  two  facts  agree  in  general  with  Hehner’s  conclusions 
that  25  cc.  (1.01  per  cent)  of  picric  acid  should  be  used  and  that 
an  additional  amount  produces  no  different  effect.  However, 
mention  should  be  made  that  in  our  case  where  21  cc.  of  the  1.2 
per  cent  picric  acid,  which  is  the  equivalent  of  25  cc.  of  a 1.01 
per  cent  solution  was  used,  no  decided  change  was  produced. 
This  fact  is  shown  in  the  data  in  Table  4.  Further,  while  the 
additional  amount  of  picric  acid  seems  in  general  to  cause  a deeper 
color,  the  authors  wish  to  emphasize  the  fact  that  this  difference 
is  by  no  means  as  great  as  Hehner  states  it  to  be  in  his  paper. 
After  calculating  the  data,  obtained  by  using  30  cc.  of  picric  acid 
and  10  cc.  of  alkali,  it  will  be  seen  that  the  combined  percentages 
of  creatinin  and  creatin  in  the  six  different  samples  of  beef  extract 
reported  in  Tables  2,  4,  and  7 amount  to  4.15,  4.11,  6.71,  3.80, 
1.63,  5.24  and  6.46.  These  samples  correspond,  respectively,  to 
those  for  Laboratory  Nos.  2278a,  2278c,  2306,  2307,  2308,  2309 
and  2310.  When  these  percentages  are  compared  with  those 
reported  in  the  previous  paper  which  ranged  from  1.38  to  6.56 
per  cent,  it  can  be  stated  that  the  data  do  not  differ  materially 
in  the  two  cases,  and  in  as  much  as  the  samples  used  for  this 
work  are  both  representative  of  those  reported  formerly  and  also 
of  Hehner’s  the  evidence  seems  to  indicate  that  Hehner’s  results 
which  varied  from  10  to  12  per  cent  for  the  combined  creatinin 
and  creatin  were  entirely  too  high. 

From  the  above  consideration  of  the  data,  it  is  evident  that 
in  the  majority  of  cases  the  method  gives  higher  results  for  the 
converted  creatin  in  meat  extracts,  meat  and  pure  creatin  when 
the  quantity  of  picric  acid  (1.2  per  cent)  is  increased  from  15  to 
30  cc.,  although  several  instances  are  reported  where  the  smaller 
amount  of  acid  served  equally  well.  No  definite  explanation  can 
be  given  at  the  present  time  for  this  apparent  anomaly.  The 


Chemistry  of  Flesh 


5M 

tendency  seems  to  indicate  that  it  is  more  difficult  for  the  1 5 cc.  of 
picric  acid  to  overcome  the  resistance  of  the  resulting  converted 
creatinin  than  that  of  the  preformed  creatinin  which  shows  that 
the  former  must  exist  in  a different  condition  than  the  latter. 
The  amount  of  hydrochloric  acid  used  in  dehydrating  the  creatin 
does  not  seem  to  influence  the  results  as  is  shown  in  Experiment 
10  which  relates  to  the  pure  creatin.  Here,  some  solutions  had 
90  cc.  of  hydrochloric  acid  and  others  50  cc.,  yet  the  general  effect 
of  the  picric  acid  was  the  same  in  each  case.  Jaffe,1  states  that 
by  using  the  zinc  chloride  method  his  maximum  yield  was  94.83 
per  cent  and  adds  that  this  seems  to  show  that  the  creatinin 
resulting  from  converted  creatin  is  broken  down  to  some  extent 
by  the  strong  acid.  In  general  the  results  reported  in  this  paper 
confirm  Jaffe’s  conclusion.  Benedict  and  Meyers2  in  using  Folin 
method  with  creatin  obtained  from  96  to  98.9  per  cent,  while  the 
data  here  reported  show  a variation  in  six  determinations  of 
from  94.2  per  cent  to  95.8  per  cent,  and  in  one  test  the  result  was 
99.98  per  cent.  These  facts,  however,  do  not  necessarily  reflect 
upon  the  Folin  colorimetric  method  as  applied  to  urine,  meat  and 
meat  extract.  Since  normal  urine  contains  no  creatin,  meats  only 
0.44  per  cent,  and  meat  extracts  from  1 to  6 per  cent,  it  will  be 
seen  that  a yield  of  95  per  cent  is  sufficiently  accurate  for  all 
practical  purposes.  Further,  in  as  much  as  the  modified  method 
seems  to  give  uniform  results  throughout,  the  data  should  be 
comparable  in  all  cases  and  be  of  extreme  value  in  giving  impor- 
tant information  as  to  the  quantity  of  these  extractives  in  meat 
products. 

OUTLINE  OF  METHOD  AS  NOW  USED  IN  THIS  LABORATORY. 

The  following  brief  outline  of  the  method  as  now  used  is  given : 
For  the  preformed  creatinin,  transfer  aliquot  portions  of  the 
sample  solution  to  500  cc.  measuring  flasks,  add  15  cc.  of  a 1.2  per 
cent  picric  acid  solution,  mix,  add  10  cc.  of  a 10  per  cent  sodium 
hydroxide  solution,  shake  thoroughly,  and  allow  the  mixture 
to  stand  5 minutes  and  then  dilute  to  the  mark  at  once  and  after 
mixing,  compare  the  depth  of  the  color  of  the  solutions  with  that 

1 Zeitschr.  f.  physiol.  Chem.,  xlviii,  p.  436,  1906. 

2 Amer.  Journ.  of  Physiol xviii,  p.  4 1907. 


A.  D.  Emmett  and  H.  S.  Grindley  515 

of  a half-normal  bichromate  solution  set  at  8 mm.  According  to 
Folin,  the  correct  reading  in  millimeters  of  the  colorimeter 
divided  into  81  gives  the  number  of  milligrams  of  creatinin 
contained  in  the  portion  of  the  solution  taken  for  the  treatment 
with  picric  acid  and  sodium  hydroxide.  In  other  words,  10 
milligrams  of  pure  creatinin  after  the  addition  of  the  picric 
acid  and  the  alkali  and  dilution  to  500  cc.,  gives  a reading  of 
8.1  mm.  when  compared  with  8 mm.  of  f potassium  bichromate 
solution  (24.54  grams  per  liter).  For  the  combined  creatinin, 
transfer  aliquot  portions  of  the  sample  solution  to  beakers, 
if  the  quantity  is  more  than  10  cc.,  or  to  100  cc.  measuring 
flasks,  if  the  quantity  is  10  cc.  or  less.  In  the  former  instance, 
evaporate  the  solution  on  the  water-bath  to  10  cc.  In  either  case 
make  the  volume  of  the  liquid  up  10  cc.,  if  necessary,  and  add  10 
cc.  of  normal  hydrochloric  acid.  Rotate  the  vessels  to  mix  the 
liquids.  Transfer  the  acid  solutions  to  an  autoclave  and  heat 
them  at  a temperature  of  117  to  1190  C.  for  30  minutes.  After 
removal,  cool  and  dilute  to  the  mark.  If  beakers  were  used, 
transfer  the  contents  to  100  cc.  measuring  flasks  and  dilute.  To 
aliquot  portions  of  the  converted  creatinin  solution  add,  in  500  cc. 
flasks,  30  cc.  of  1.2  per  cent  picric  acid,  shake  and  then  add  10  cc. 
of  the  10  per  cent  sodium  hydroxide.  Mix  thoroughly  and  after 
standing  exactly  5 minutes  dilute,  and  read  the  depth  of  color 
of  the  solution.  In  order  to  convert  milligrams  of  creatinin 
into  creatin  multiply  by  the  factor  1.16. 

It  was  found  that  by  using  a black  cloth,  to  shut  out  the  sur- 
rounding light  from  the  eyepiece  of  the  instrument,  the  colors 
appeared  more  distinctly,  and  that  the  comparison  could  be 
made  more  accurately  and  rapidly  and  with  less  strain  on  the 
eye. 

CONCLUSIONS. 

From  this  study  upon  meat  extracts  and  meat,  the  following 
conclusions  can  be  made  in  regard  to  the  applicability  of  the 
Folin  method  for  determining  creatinin  and  creatin: 

(a)  That  an  increase  in  the  quantity  of  picric  acid,  according 
to  Hehner’s  suggestion  causes  no  difference  in  the  so-called 
original  creatinin  determinations;  but  it  generally  does  produce 
an  appreciable  difference  when  the  converted  creatin  is  also 


Chemistry  of  Flesh 


5l6 

present,  and,  further,  that  the  quantity  of  picric  acid  (1.2  per 
cent)  recommended  for  use  in  meat  extract,  meat  and  urine 
should  be  left  at  15  cc.  for  the  original  creatinin  determinations 
and  be  increased  to  30  cc.  for  the  dehydrated  creatinin. 

(b)  That  in  the  determination  of  the  original  creatinin,  the 
use  of  a small  or  large  amount  of  10  per  cent  alkali  makes  almost 
no  difference,  the  5 cc.  quantity  giving  slightly  lower  results  than 
the  10  and  15  cc.  quantities;  that,  for  the  converted  creatin,  the 
previously  accepted  quantity  of  alkali,  10  cc.,  gives  better  results 
than  5 cc.  and  equally  as  good  results  as  the  large  excess,  1 5 cc. ; 
and  further,  that  these  facts  are  contrary  to  those  found  by  Heh- 
ner  who  states  that  a quite  small  amount  of  alkali  gives  better 
results  than  a large  quantity  which  he  maintains  diminishes 
the  depth  of  color. 

(c)  That  the  data  reported  are  representative  of  the  percent- 
ages of  creatinin  and  creatin  in  meats  and  meat  extracts,  being 
practically  the  same  for  the  combined  extractives  as  those  pre- 
viously published,  0.45  per  cent  for  the  former  and  1.4  to  6.5 
per  cent  for  the  latter,  whereas  Hehner  found  the  total  percent- 
age of  creatinin  and  creatin  in  meat  extracts  to  be  10  to  12  per 
cent. 

(d)  That  the  Folin  method  when  properly  modified  is  as 
applicable  to  meat  extracts  and  meats  as  it  is  to  urine,  and  that 
it  gives  reliable  and  concordant  results  in  the  hands  of  different 
analysts  of  this  laboratory. 

The  authors  wish  to  acknowledge  their  appreciation  of  the 
assistance  rendered  by  Messrs,  H.  H.  Mitchell  and  D.  L.  Weather- 
head. 


ERRATA,  VOLUME  III. 

Page  85,  line  26,  for  exclusive  read  extensive. 

Page  90,  line  7,  for  chloride  read  hydroxide. 

Page  180,  Nos.  (3),  (4),  (5)  and  (6)  of  table  of  acetone  determinations 
should  read,  “20  cc.  acetone  solution  + o cc.  H20,  etc.” 


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